Class switch recombination is a pathway important for proper immunity, and misregulation is associated with specific proto-oncogene translocation events that cause lymphoma. In response to pathogenic microorganisms, B cells induce immunoglobulin isotype (class) switching, which replaces the existing antibody constant region for a downstream region, permanently deleting the intervening sequence and changing antigen clearance properties of the antibody. This recombination reaction requires the participation of conserved DNA repair proteins and the transcription of "switch" regions located upstream of each constant region. The mechanistic contribution of these regions and the participation of key repair factors in class switching are not understood. This proposal is focused on clarifying the mechanism of class switch recombination by examining the role of the mismatch repair complex, MSH2/MSH6 (MutSalpha), in the pathway. Our research will build on novel activities discovered for MutSalpha, which has been shown to specifically recognize alternatively structured DNA found at the switch regions. Our results argue that MutSalpha has two cellular functions relevant to class switch recombination and cancer, one is aligned with mismatch repair and the other is associated with recombination. We will define how MutSalpha interacts with substrates presented at the expressed immunoglobulin switch regions, illuminating in the process mechanisms of DNA recombination and the cellular functions of MutSalpha. This will be accomplished in two specific aims. (1) We will use site-directed mutagenesis and photo-crosslinking to determine the alternative structure-binding domain of human MutSalpha. Results will help define how MutSalpha activities are partitioned during class switch recombination. (2) We will use molecular assays to define how MutSalpha interacts with heteroduplex and recombination targets formed at the switch regions during class switch recombination, directly testing the model that MutSalpha plays a structure stabilization role that may be important for recruitment of recombination and repair factors. These aims are designed to provide a mechanistic understanding of early steps in the class switch recombination pathway by defining interactions of MutSalpha with the transcribed switch regions, which will provide new insights into the function of MutSalpha in modulating the immune response, regulating proto-oncogene translocation events, and maintaining genomic stability. PUBLIC HEALTH RELEVANCE Establishing the function of MutS1 in class switch recombination will clarify an important immune system pathway, and help define a mechanism of gene rearrangement that causes Burkitt's lymphoma. In addition, MutS1 is a highly conserved DNA repair factor, so understanding how it functions elucidates genome maintenance pathways important for preventing mutagenesis and cancer.